What are the new molecules for USP7 inhibitors?

Introduction to USP7
Ubiquitin‐specific protease 7 (USP7) is a key member of the deubiquitinating enzyme (DUB) family that plays a central role in regulating the ubiquitin–proteasome system. By removing ubiquitin moieties from specific target proteins, USP7 controls the stability and activity of numerous cellular regulators involved in tumor suppression, cell cycle progression, DNA damage repair, and immune responses. Its intrinsic multi‐domain architecture—with an N‐terminal TRAF-like domain responsible for substrate recognition and a catalytic domain whose conformation changes upon binding to ubiquitin—confers a tightly regulated mechanism that balances protein stability and degradation.

Role and Function of USP7
USP7 exerts its activity by interacting with, and deubiquitinating, substrates such as p53, MDM2, PTEN, FOXO, and even components of the DDR machinery. Under basal conditions, USP7 participates in the fine tuning of the p53–MDM2 axis by stabilizing both p53 and its negative regulator MDM2, although the net effect in many cancer cells favors p53 destabilization by preferentially deubiquitinating MDM2. Structural studies have demonstrated that USP7 exists in an inactive conformation until binding of ubiquitin induces a rearrangement of the catalytic residues, rendering the enzyme active. This unique switch – dependent on a conformational change – has generated substantial interest because it implies the possibility of selective inhibition via nontraditional binding pockets other than the highly conserved catalytic site.

Importance in Disease Pathology
Dysregulation of USP7 has been linked to a wide range of diseases, most notably in various forms of cancer. Overexpression or hyperactivity of USP7 is often correlated with tumor progression, chemoresistance, and poor prognosis because it affects the stability of proteins that govern cell survival and apoptosis. For instance, USP7-mediated stabilization of MDM2 leads to proteasomal degradation of p53 in multiple tumor types, thereby compromising the tumor suppressive functions of p53. Beyond its implication in the p53–MDM2 pathway, USP7 modulates proteins involved in DNA repair pathways, epigenetic regulation, and even immuno-oncology circuits. Its central role in these processes has made USP7 an attractive drug target for cancer therapy and has spurred a vigorous discovery program aimed at identifying novel small-molecule inhibitors with improved selectivity and potency.

Current Landscape of USP7 Inhibitors
The landscape of USP7 inhibitors has evolved over the years as researchers have strived to target the enzyme’s catalytic machinery and allosteric regulatory sites. Preclinical studies using chemical inhibitors have demonstrated that interrupting USP7 activity can restore p53 function and propel tumor cell death.

Existing USP7 Inhibitors
Historically, several small molecules have been reported to inhibit USP7, including HBX41,108, HBX19,818, P5091, and P22077. These inhibitors were discovered primarily through high-throughput screening (HTS) campaigns and have been applied in various cellular model systems. For example, P5091 and its optimized derivative P22077 have been used extensively in both in vitro and in vivo studies to demonstrate the antitumor potential of USP7 inhibition in multiple myeloma, neuroblastoma, and colon cancer models. Moreover, GNE-6640 and GNE-6776 were identified using nuclear magnetic resonance (NMR)-based screening approaches, which provided novel insights into interfering with the binding of ubiquitin to USP7 and destabilize substrates such as MDM2. Despite their promising results, most of these early molecules exhibit limitations in terms of selectivity, off-target activities, and suboptimal pharmacokinetic properties.

Limitations of Current Inhibitors
The existing USP7 inhibitors face several common shortcomings. First, many of the early-generation inhibitors predominantly target the catalytic domain indiscriminately, which poses the challenge of affecting multiple deubiquitinating enzymes because of the structural similarities within the USP family. Second, off-target effects and limited selectivity have hampered their clinical translation, particularly because the ubiquitin-proteasome system is intricately involved in a plethora of biological processes. In addition, some of these molecules are chemically reactive and may form covalent adducts in a promiscuous fashion, leading to toxicity issues upon systemic administration. Finally, the first-generation inhibitors often lack favorable in vivo stability and bioavailability, which further reduces their potential as clinical candidates. This has increased the impetus for the discovery of new USP7 inhibitors with improved target selectivity, better physicochemical properties, and an allosteric mode of action that circumvents the canonical catalytic site.

Discovery of New USP7 Inhibitors
Recent advances in fragment-based screening, structure-based drug design, and computational modeling have revolutionized the discovery of novel USP7 inhibitors. With refined screening technologies and sophisticated medicinal chemistry techniques, new molecules are now emerging that can modulate USP7 activity with high potency and selectivity.

Recent Discoveries and Developments
A number of recent studies have significantly advanced our understanding of USP7 inhibitor development. The application of fragment-based and NMR-driven screening methods has led to the discovery of noncovalent, reversible, and allosteric inhibitors that bind outside the conventional active site. Researchers using SPR screening combined with structural elucidation and X-ray crystallography were able to identify hit fragments that interact with a novel allosteric “palm” region of USP7. This breakthrough approach revealed not only that USP7 has additional druggable pockets but also that high-affinity inhibitors can be designed to modulate the enzyme’s activity by stabilizing its inactive conformation.

For example, two selective inhibitors, GNE-6640 and GNE-6776, were discovered using NMR-based screening in a study by Kategaya et al. These molecules bind noncovalently within a region that interferes with the positioning of ubiquitin, thereby competitively inhibiting USP7’s enzymatic activity. In parallel, researchers employed fragment-based screening methodologies that resulted in the optimization of initial hits by combining structural features and medicinal chemistry modifications. As a result, compounds such as compound 4 and compound 2, as reported by Gavory et al., were optimized to achieve submicromolar potency; compound 4, in particular, emerged as one of the most potent allosteric inhibitors with an IC₅₀ in the low nanomolar range (~6 nM), underlining the benefit of an allosteric, fragment-based approach over traditional catalytic inhibition.

Another major development is the identification of an irreversible inhibitor derivative named XL177A, an analogue of XL188. XL177A covalently binds USP7 with sub-nanomolar potency and displays remarkable selectivity. Its irreversible binding is associated with a unique chemical warhead that modifies the catalytic cysteine upon stabilizing the inactive state of USP7. This compound’s development illustrates the feasibility of selective, covalent modulation of USP7 and underscores its potential for therapeutic use in cancer lines where p53 mutational status is an important factor.

Compound 41 is another novel small-molecule inhibitor recently reported that distinguishes itself by being reversible, highly potent, and orally bioavailable. It was crafted by optimization of an initial benzofuran-amide scaffold, which was redesigned to create an ether series. The key structural modification involved replacing ether-linked amines with carbon-linked morpholines through computer-aided free energy perturbation (FEP+) calculations. The result is a molecule that not only exhibits robust binding affinity toward USP7 but also demonstrates in vivo antitumor activity in xenograft models of multiple myeloma, suppressing tumor growth in both p53 wild-type and mutant cell lines.

In addition, a new series of USP7 inhibitors based on the pyrimidinone scaffold have been reported. These molecules were generated through structure-guided design, scaffold-hopping, and hybridization strategies stemming from fragment-based hits. Although the study focused on establishing structure-activity relationships (SAR), the identified series provides a promising platform to develop compounds suitable for in vivo evaluation in cancer treatment models.

Other advances include the identification of natural product-based inhibitors, such as spongiacidin C, a pyrrole alkaloid obtained from marine sponge Stylissa massa that has shown selectivity for USP7 in biochemical assays. While natural product-based inhibitors often require further validation in cellular contexts, they represent an emerging class that may be further optimized for improved efficacy.

Novel Molecules and Their Mechanisms
The novel USP7 inhibitors uncovered in the recent literature can be divided broadly into noncovalent allosteric inhibitors, irreversible covalent inhibitors, and natural product scaffolds. Each of these groups brings distinct mechanisms for modulating the activity of USP7.

• Noncovalent allosteric inhibitors
The new noncovalent molecules such as GNE-6640, GNE-6776, and the compounds developed through fragment-based approaches (compound 2 and compound 4) operate by binding to a newly characterized allosteric site on USP7. By interacting with the “palm” region of the catalytic domain, these inhibitors hinder the proper positioning of ubiquitin necessary for catalytic activity. The mechanism is characterized by competitive inhibition of ubiquitin binding rather than direct disruption of the catalytic cysteine’s reactivity. The binding leads to a conformational shift that maintains USP7 in an inactive state, thereby reducing the stabilization of oncogenic substrates like MDM2. This allosteric inhibition strategy not only enhances selectivity but also minimizes off-target effects when compared to inhibitors that target the highly conserved catalytic cysteine.

• Irreversible covalent inhibitors
XL177A is the standout compound in the irreversible inhibitor class. As a covalent modifier, XL177A attaches to the catalytic cysteine residue via its electrophilic warhead, thereby permanently disabling USP7’s deubiquitinating function. This irreversible bond formation results in sustained inhibition even in the presence of high intracellular concentrations of substrate, leading to effective downregulation of oncogenic pathways such as the p53–MDM2 axis. Importantly, the selective and irreversible nature of XL177A has been associated with a potent antiproliferative effect in tumor cell lines, especially those with mutant p53 where conventional strategies may fail.

• Optimized reversible inhibitors with improved bioavailability
Compound 41, through its elegantly modified benzofuran and ether-morpholine scaffold, represents a crucial advancement in achieving oral bioavailability and high selectivity. The use of computational methods such as FEP+ allowed researchers to fine-tune the compound’s binding energy and pharmacokinetic properties. Mechanistically, compound 41 binds reversibly to USP7, leading to a decrease in the levels of substrates like MDM2, and subsequent activation of p53-dependent and p53-independent cell death pathways. Its efficacy in vivo emphasizes its potential for clinical development as a therapeutic agent that can circumvent some of the limitations observed with earlier molecules.

• Pyrimidinone-based inhibitors
The pyrimidinone scaffold-based series also represents a novel class that emerged from recent medicinal chemistry efforts. Initial hits in this series were obtained via fragment screening and subsequently optimized by structural modifications in response to X-ray crystallographic data. The unique interactions these compounds establish in the USP7 binding pocket point to new opportunities in modulating enzyme activity through nontraditional binding modes, expanding the chemical space available for potent and selective inhibition.

• Natural product-based inhibitors
Natural sources continue to offer structurally diverse compounds as starting points for drug discovery, and spongiacidin C is a prime example. Despite demonstrating high selectivity in biochemical assays, its efficacy in cellular contexts needs further exploration. The reported activity against USP7, however, supports the notion that nature-derived scaffolds can serve as innovative leads that may eventually complement or inspire synthetic modifications to enhance their pharmacological profiles.

Overall, these novel molecules share a common objective: they are designed to exploit the unique structural and regulatory features of USP7. By targeting novel binding pockets and utilizing both reversible and irreversible interactions, these compounds are structured to offer more favorable selectivity profiles, potency, and in some cases, oral bioavailability. Such attributes hold promise for improved therapeutic indices and reduced adverse effects in cancer treatment applications.

Challenges and Future Directions
While significant progress has been made in the identification and optimization of USP7 inhibitors, several challenges persist in translating these discoveries into clinically viable therapies. Strategic approaches to overcome these hurdles focus on enhanced selectivity, understanding off-target interactions, and developing more robust preclinical models to investigate complex tumor suppressor circuitry.

Challenges in Developing USP7 Inhibitors
One major challenge lies in the inherent complexity of USP7 itself. Given its multifaceted roles—ranging from DNA damage repair to epigenetic regulation—the inhibition of USP7 must be finely tuned to avoid disrupting essential cellular functions in noncancerous tissues. For instance, complete abrogation of USP7 activity through indiscriminate catalytic inhibition might trigger toxicity issues given the enzyme’s pivotal role in maintaining cellular protein homeostasis. Early compounds such as P5091 and HBX-based inhibitors, although valuable for proof-of-concept studies, have shown limited specificity due to their broad reactivity with similar cysteine proteases.

Another challenge is related to the evolution of drug resistance. Tumors exhibiting adaptive resistance mechanisms or compensatory pathway activations can undermine the efficacy of USP7 inhibitors. Such resistance may be inherent in tumors that rely on multiple regulatory nodes for survival and proliferation, calling for inhibitors that are effective regardless of p53 mutation status and that can overcome alternative survival pathways.

Furthermore, achieving favorable pharmacokinetic properties has been a consistent difficulty. The balance between high potency and the necessary bioavailability in vivo continues to be a delicate one, with many potential leads failing to translate from in vitro studies to animal models due to metabolism, poor absorption, or tissue-specific toxicity.

Last but not least, the identification of optimal binding sites, whether through the catalytic site or allosteric regions, requires a comprehensive understanding of the dynamic conformation of USP7. This is complicated by the fact that USP7’s active conformation is induced only upon ubiquitin binding, making it challenging to identify states that are most amenable to selective modulation.

Future Research and Development Opportunities
Given these challenges, several promising avenues for future research have emerged. First, the discovery of allosteric sites provides new therapeutic opportunities that may allow inhibition of USP7 with higher specificity and fewer off-target effects. Future studies may further explore and validate these novel binding pockets using integrated computational, biophysical, and structural biology methods. The use of fragment-based screening combined with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and cryo-EM can provide additional insights into transient conformational states that are otherwise elusive.

Second, leveraging machine learning and artificial intelligence approaches in drug design can accelerate the optimization of lead compounds. Advanced models now integrate chemical, biological, and structural data to predict drug-target interactions, improving hit rates and further refining the pharmacokinetic profiles of promising molecules. Such computational strategies are being applied to design novel USP7 inhibitors that possess both high target specificity and favorable in vivo profiles.

Third, combinatorial drug therapies may offer enhanced efficacy by simultaneously targeting USP7 and related signaling pathways. For instance, combining USP7 inhibitors with CDK1 inhibitors or other agents that promote premature cell cycle progression has been proposed to achieve synergistic anticancer effects. This combination strategy not only enhances tumor cell death but may also help overcome resistance mechanisms.

Additionally, future preclinical studies must strive to develop more comprehensive models that encapsulate the complexity of USP7 function in vivo. The development of genetically engineered mouse models (GEMMs) that conditionally knock out or express mutant forms of USP7 could help elucidate tissue-specific functions and toxicity profiles. These models are crucial for evaluating the safety and efficacy of both reversible and irreversible inhibitors such as compound 41 and XL177A.

Natural product-based screening remains a compelling opportunity as well. The structural diversity found in natural products, exemplified by spongiacidin C, can be harnessed and optimized through semi-synthetic modifications to yield inhibitors with improved selectivity. Such hybrids, bridging natural scaffolds with synthetic chemistries, may open new vistas in the development of USP7 inhibitors.

Finally, there is a clear opportunity for advancing clinical trials for these new molecules. While a number of second-generation USP7 inhibitors show promise in preclinical studies, their transition into clinical evaluation requires robust pharmacology and toxicology studies. The insights obtained from detailed structure-activity relationship studies and interdisciplinary research will guide dosage optimization, patient selection, and combination strategies in clinical settings. Maintaining a close dialogue between medicinal chemists, structural biologists, and clinical oncologists will be essential for accelerating the bench-to-bedside translation of these novel compounds.

Conclusion
In summary, new molecules for USP7 inhibitors have emerged that significantly improve upon the limitations of earlier compounds. Early inhibitors such as HBX41,108 and P5091 laid the groundwork by demonstrating that USP7 is a viable target in cancer therapy. However, their broad reactivity, lack of selectivity, and suboptimal pharmacokinetic profiles necessitated the exploration of novel approaches. Recent breakthroughs have resulted in the identification of innovative compounds including noncovalent allosteric inhibitors (e.g., GNE-6640, GNE-6776, compound 2, and compound 4), the irreversible inhibitor XL177A, and the newly optimized reversible molecule compound 41. These compounds, discovered via advanced methodologies such as fragment-based screening, NMR-based assays, and structure-guided design, exploit unique binding sites outside the conventional catalytic pocket and provide superior potency, selectivity, bioavailability, and even oral administration potential.

Though the development of novel USP7 inhibitors presents several challenges—including potential toxicity due to the enzyme’s pleiotropic functions, the need to overcome adaptive resistance mechanisms, and the difficulty in achieving sustained in vivo efficacy—ongoing research is focused on addressing these issues through allosteric modulation, advanced computational methods, and combination therapy approaches. As these innovative compounds move forward in preclinical and eventually clinical stages, they represent a significant evolution in our ability to target an enzyme that is central to oncogenesis, particularly in tumors with deregulated p53–MDM2 dynamics.

To conclude, the discovery and development of new USP7 inhibitors such as XL177A, compound 41, GNE-6640/GNE-6776, and the fragment-derived compounds (compound 2 and compound 4) offer promising therapeutic avenues for cancer treatment. These new molecules are designed not only to inhibit USP7 activity more selectively and potently than previous inhibitors but also to overcome the limitations of early candidates by leveraging structure-based insights and innovative screening approaches. They set the stage for the next generation of targeted cancer therapeutics, potentially leading to improved patient outcomes and a more effective modulation of the ubiquitin–proteasome system in disease pathology. The integration of diverse discovery strategies and computational methodologies, along with rigorous preclinical validation, will be key in overcoming the current challenges and unlocking the full potential of USP7 inhibition in clinical oncology.